I. Field of the Invention
The present invention relates generally to the fields of cellular and molecular biology. More specifically, the present invention relates to antagonism of TGF-β receptors by variants of the TGF-β superfamily of ligands and uses thereof.
II. Description of Related Art
The transforming growth factor β (TGF-β) superfamily comprises over 30 secreted ligands in humans that control cell proliferation, cell death, metabolism, homeostasis, differentiation, tissue development, immune responses, angiogenesis, wound repair, endocrine function and many other physiologic processes. Members of this superfamily include TGF-β, activin, bone morphogenetic protein (BMP), Growth and Differentiation Factor (GDF) and nodal-related families. Disruption or dysregulated activity of TGF-β superfamily members is associated with multiple pathological states resulting from processes including aberrant cellular differentiation, proliferation and/or metabolism.
TGF-β superfamily members share a distinct structural framework known as the cystine knot scaffold. Activins adopt this prototypical disulfide-linked dimeric structure and consist of two β chains. Although there are several activin β subunit genes and an extensive array of possible β-β dimers, only βA-βA (activin-A), βA-βB (activin-AB) and βB-βB (activin-B) have been isolated as dimeric proteins and shown to be biologically active. Similar to other TGF-β superfamily members, activins derive from large precursor proteins consisting of an N-terminal prodomain and a C-terminal β subunit domain that is released following proteolysis. In the case of activins the prodomain aids in proper folding, disulfide bond formation and dimerization of the β subunit domains after which the mature active β subunit dimers are cleaved and released. In the case of other TGF-β superfamily members, such as the TGF-β isoforms and myostatin (GDF8), however, the prodomain remains bound to the mature ligand to inhibit its activity.
Signaling via Receptor Serine Kinases—Activins and other TGF-β superfamily members exert their biological effects by interacting with two types of cell surface transmembrane receptors (type I and type II receptors) with intrinsic serine/threonine kinase activities, called receptor serine kinases (RSKs). The type II activin receptor (ActRII) was the first RSK to be cloned and led to the cloning of four additional type II RSKs and seven type I RSKs in human. The type I RSKs are referred to as ALK1 to ALK7, for Activin receptor-Like Kinases. The receptor activation mechanism for all TGF-β ligands involves both receptor types. This was first established for TGF-β that was shown to bind its type II receptor (TβRII) leading to the recruitment, phosphorylation and activation of its type I receptor (ALK5) followed by activation of intracellular signaling. A similar mechanism of ligand-mediated receptor assembly and type I receptor phosphorylation was demonstrated for activin-A, although activin-A binds ActRII or ActRIIB followed by recruitment, phosphorylation and activation of its type I receptor ALK4. Considerable promiscuity exists in the interactions between TGF-β ligands and receptors since there are over 30 ligands in human signaling via only five type II receptors and seven type I receptors. Activin type II receptors are particularly promiscuous and have been shown to mediate signaling of at least 12 TGF-β ligands including activins, myostatin (GDF8), bone morphogenetic proteins (BMPs) and nodal.
Regulation of Transcriptional Responses via Smads—Based upon genetic studies in Drosophila and Caenorhabditis elegans, a group of proteins now called Smads have been found to transduce signals from receptor serine kinases and mediate regulation of target gene transcription by activin-A and other TGF-β superfamily members. Structural and functional considerations allow subdivision of Smads into three subfamilies: pathway-specific, common mediator, and inhibitory Smads. Ligand/receptor assembly and activin receptor-like kinase (ALK) phosphorylation triggers a transient ALK/pathway-specific Smad association during which the ALK phosphorylates the Smad on its last two serine residues in the C terminal SSXS motif. Activin-A and TGF-β signals are mediated by the pathway-specific Smads, Smad2 and Smad3, while BMP signals are mediated by Smads 1, 5 and 8. Once activated, pathway-specific Smads form hetero-oligomeric complexes with the common mediator, Smad4. These Smad complexes then translocate into the nucleus and interact directly with DNA and/or with cell-type specific co-activator or co-repressor proteins leading to the activation or repression of target genes. Two vertebrate inhibitory Smads have been identified, Smad 6 and 7, which lack the C-terminal SSXS motif found in the pathway specific Smads. Smad 6 and 7 are inhibitors of Smad signaling and bind to ALKs to prevent phosphorylation and activation of the pathway-specific Smads.
Current methods for antagonizing the activation of TGF-β receptors typically use follistatin or other proteins or agents that compete with the TGF-β receptors for binding, in essence sequestering the ligand away from the receptor. Additional compositions and methods for the use and development of high affinity selective modulators and antagonists of the receptors for TGF-β superfamily of ligands, such as type II activin receptors, are needed.